This invention relates to the field of compositions and methods for inhibiting the enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase in geranium thereby prolonging the shelf-life of cut flowers as well as reducing leaf yellowing and petal abscission during shipping and storage. Specifically, identification of the PHSacc-25 gene of geranium which may be used individually or in combination with previously identified ACC synthase genes for genetic modification of geranium.
A variety of factors cause wilting and natural abscission in flowers, particularly after a cutting of the plant or when flowers have been removed from the plant. Such factors include increased oxygen levels, wounding, chemical stress, and the plant""s own production of ethylene. Of these factors, the plant""s production of ethylene, has been shown to play a key role in natural senescence, the degenerative process which generally leads to controlled cell death in plants, but also in the degradation of flowers after they have been cut.
Ethylene, in all higher plants, is important to plant growth and development with respect to seed germination, seedling growth, flowering, and senescence (Abeles, F. B. et al. (1992), In: Ethylene in Plant Biology, Academic Press, New York, pp. 285-291; Morgan, M. E. Saltveit, J.R., xe2x80x9cIntroduction and Historical Perspectivesxe2x80x9d, Ethylene In Plant Biology, (1991), pp.1-13; Yang, S. F., et al., xe2x80x9cEthylene Biosynthesis and its Regulation in Higher Plantsxe2x80x9d, Plant Physiology Annual Review (1984), pp. 155-189). Ethylene production in plants can also be associated with trauma induced by mechanical wounding, chemicals, stress (such as produced by temperature and water amount variations), and by disease. Hormones can also stimulate ethylene production. Such ethylene, also sometimes called xe2x80x9cstress ethylenexe2x80x9d, can be an important factor in storage effectiveness for plants. Moreover, exposure of plant tissue to a small amount of ethylene often may be associated with increased production of ethylene by other adjacent plants. This autocatalytic effect may be often associated with losses in marketability of plant material during storage and transportation (Abeles et al., supra; Yang et al., supra).
The ethylene biosynthetic pathway in plants was established by Adams and Yang (Adams, D. O., et al., xe2x80x9cEthylene biosynthesis: Identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylenexe2x80x9d, Proc. Nat""l Acad Sci USA 76: 170-174). The first step involves the formation of S-adenosyl-L-methionine (AdoMet) from methionine by S-adenosyl-L-methionine synthetase. AdoMet is then converted to 1-aminocyclopropane-1-carboxylate (ACC), the direct precursor of ethylene in higher plants. This conversion is catalyzed by ACC synthase (S-adenosyl-L-methionine methyl thioadenosine-lyase, EC4.4.1.14), the rate limiting step in the ethylene biosynthetic pathway. (See also Kionka, C., et al., xe2x80x9cThe enzymatic Malonylation of 1-aminocyclopropane-1-carboxylic Acid in Homogenates of Mung Bean Hypocotylsxe2x80x9d, Planta 162: 226-235, (1984); Amrhein, N., et al., xe2x80x9cIdentification of a Major Metabolite of the Ethylene Precursor 1-Aminocyclopropane-1-carboxylic Acid in Higher Plants.xe2x80x9d, Naturwissenschaften 68: 619-620 (1981); Hoffman, N. E., et al., xe2x80x9cIdentification of a 1-(malonylamino)cyclopropane 1-carboxylic acid as a major conjugate of 1-aminocyclopropane-1-carboxylic acid, an ethylene precursor in Higher Plantsxe2x80x9d, (1982), Biochem. Biophys. Res. Common. vol. 104, no. 2, pp.765-770.
Knowledge of the biosynthetic pathway for ethylene formation has been fundamental in developing strategies for inhibiting ethylene production in plants. One approach has been to use chemical inhibitors to inhibit the synthesis or activity of ethylene, two of the most common being aminoethoxyvinylglycine and aminooxyacetic acid (Rand, R. R., xe2x80x9cChemistry and Enzymology of KCat Inhibitorsxe2x80x9d (1974), Science 185, pp. 320-324) and in Ethylene in Plant Biology, (Abeles, F. B., et. al., Ethylene in Plant Biology., (1992), pp. 285-291). However, chemical methods find limited use because such methods are expensive and the beneficial effect they provide is generally only short-lived.
A second approach has been to over express ACC deaminase, an enzyme which metabolizes ACC, thereby eliminating an intermediate in the biosynthesis of ethylene (Klee, et al., xe2x80x9cControl of Ethylene Synthesis by Expression of a bacterial enzyme in Trangenic Tomato plants.xe2x80x9d, (1991), Plant Cell 3: 1187-1193) (See also Theologis, A., et al., xe2x80x9cModifying Fruit Ripening By Supressing Gene Expressionxe2x80x9d, Cellular and Molecular Aspects of the Plant Hormone Ethylene, (1993), pp. 19-23). Because ACC deaminase is a bacterial enzyme, it is heterologous, and thus, external to the plant. Thus, it is unlikely that this approach will yield a modification that will be stable from generation to generation.
Yet another approach involves attempts to genetically inhibit the production of the enzymes involved in the biosynthesis of ethylene or to inhibit the biosynthesis of the enzymes directly. This approach has the advantage of not only altering the way the plant itself functions irrespective of external factors but also of presenting a system which reproduces itself, that is, the altered plant""s progeny will have the same altered properties for generations to come.
Initial efforts to better understand the enzymes which catalyze the reactions in the biosynthesis of ethylene have involved the identification and characterization of the genes encoding for AdoMet synthetase, ACC synthase, and ACC oxidase (See also Kende, H. et al., xe2x80x9cEthylene Biosynthesis: Annual Review of Plant Physiologyxe2x80x9d, Plant Molecular Biology, (1993), pp. 283-307). Some of the genes encoding for ACC synthase have been identified for a number of plants. For instance, ACC synthase sequences have been identified for zucchini (Sato, et al., xe2x80x9cCloning the mRNA encoding 1-aminocyclopropane-1-carboxylate synthase, the key enzyme for ethylene biosynthesis in plantsxe2x80x9d, Proceedings of the National Academy of Sciences, (1989), pp. 6621-6625), winter squash (Nakajima, N., et al., xe2x80x9cMolecular Cloning and Sequence of a Complementary DNA Encoding 1-Aminocyclopropane-1-carboxylate Synthase Induced by Tissue Woundingxe2x80x9d, Plant Cell Physiology, (1990), pp. 1021-1029), tomato (Van Der Straeten, D., et al., xe2x80x9cCloning and Sequence of two different cDNAs encoding 1-aminocyclopropane-1-carboxylate Synthase in a Tomatoxe2x80x9d, Proceedings of the National Academy of Sciences, (1990), pp. 4859-4863); (Rottmann, W. H., et al., xe2x80x9cTheologis: A 1-Aminocyclopropane-i-Carboxylate Synthase in Tomato Is Encoded by a Multigene Family Whose Transcription Is Induced During Fruit and Floral Senescencexe2x80x9d, Journal of Molecular Biology, (1991), pp. 937-961), apple (Dong, J. G., et al., xe2x80x9cCloning of a cDNA Encoding 1-aminocyclopropane-1-carboxylate Synthase and Expression of its mRNA in Ripening Apple Fruitxe2x80x9d, Planta, pp. 38-45 (1991)), mung bean (Botella, J. R, et al., xe2x80x9cIdentification and Characterization of a Full-length cDNA Encoding for an Auxin-induced 1-aminocyclopropane-1-carboxylate Synthase from Etiolated Mung Bean Hypocotyl Segments and Expression of its mRNA in Reponse to Indole-3-acetic Acidxe2x80x9d, Plant Molecular Biol 20, pp. 425-436 (1992); Botella, J. R., et al., xe2x80x9cIdentification and characterization of three putative genes for 1-Aminocyclopropane-1-carboxylate Synthase from etiolated mung bean hypocotyl segmentsxe2x80x9d, Plant Mol Biol 18, pp. 793-797 (1992); Botella, J. R., et al., xe2x80x9cIdentification of two new members of the 1-Aminocyclopropane-1-carboxylate Synthase-Encoding Multigene family in mung beanxe2x80x9d, Gene 06852, pp. 249-253, (1993)); Kim W., et al., xe2x80x9cInduction of 1-aminocyclopropane-1-carboxylate Synthase mRNA by Auxin in Mung Bean Hypocotyls and Cultured Apple Shootsxe2x80x9d, (1991), Plant Physiol 98:465-471), carnation (Park, K. Y., et al., xe2x80x9cMolecular cloning of an 1-aminocyclopropane-1-carboxylate synthase from senescing carnation flower petalsxe2x80x9d, Plant Molecular Biology, (1992), Vol. 18, pp. 377-386), Arabidopsis thaliana (Liang, X., et al., xe2x80x9cThe 1-aminocyclopropane-1-carboxylate synthase gene family of Arabidopsis thalianaxe2x80x9d, Proceedings of the National Academy of Sciences, (1992), pp. 11046-11050; Van Der Straeten, et al., xe2x80x9cCloning, Genetic Mapping, and Expression Analysis of an Arabidopsis Thalinana Gene that Encodes 1-Aminocyclopropane-1-Carboxylate Synthasexe2x80x9d, Proc. Natl. Acad. Sci., (1992), pp. 9969-9973.), tobacco, rice (Zarembinski, T. I., et al., xe2x80x9cAnaerobiosis and Plant Growth Hormones Induce Two Genes Encoding 1-Aminocyclopropane-1-carboxylate Synthase in Ricexe2x80x9d, (1993) Molecular Biology of the Cell, Vol. 4: 363-373), mustard (Wen, C. M., et al., xe2x80x9cNucleotide Sequence of a cDNA Clone Encoding 1-Aminocyclopropane-1-Carboxylate Synthase in Mustardxe2x80x9d, (1993), Plant Physiol 103:1019-1020), orchid (O""Neill, et al., xe2x80x9cInterorgan regulation of Ethylene Biosynthetic Genes by Pollinationxe2x80x9d, (1993) Plant Cell 5: 419-432), broccoli (Pogson B. J., et al., xe2x80x9cDifferential Expression of Two 1-Aminocyclopropane-1-1carboxylic Acid Oxidase Genes in Broccoli after Harvestxe2x80x9d, (1995) Plant Physiol 108: 651-657), and potato (Schlagnhaufer, C. D., et al., xe2x80x9cMolecular cloning of an ozone-induced 1-aminocyclopropane-1-carboxylate synthase cDNA and its relationship with a loss of rbcS in potato (Solanum tuberosum L.)xe2x80x9d, Plant Molecular Biology, (1995), pp. 93-103).
That ACC synthase is involved in the ethylene pathway is confirmed by the fact that increased levels of ACC synthase mRNA correlate with an increased activity of ACC synthase in plants during fruit ripening and flower senescence. Similar correlation is also observed in response to exogenous signals caused either by wounding or due to treatment with hormones such as auxin, cytokinin and ethylene. Interestingly, the expression of different classes of ACC synthase occurs from a variety of signals in a many plants, e.g. four different ACC synthase genes have been shown to be differentially expressed in tomato fruit, cell cultures, and hypocotyls during ripening, wounding, and auxin treatment (Olson, D. C., et al., xe2x80x9cDifferential expression of two genes for 1-aminocyclopropane-1-carboxylate synthase in tomato fruitsxe2x80x9d, Proceedings of National Academy of Sciences, (1991), pp. 5340-5344; and Yip, W. K., et al., xe2x80x9cDifferential Accumulation of Transcripts for Four Tomato 1-aminocyclopropane-1-carboxylate Synthase Homologs under Various Conditionsxe2x80x9d, Proceedings of the National Academy of Sciences, (1992), Vol. 89, pp. 2475-2479). Differential expression of two ACC synthase genes has also been observed in winter squash during wounding or by auxin (Nakajima, N., et al., xe2x80x9cMolecular Cloning and Sequence of a Complementary DNA Encoding 1-Aminocyclopropane-1-carboxylate Synthase Induced by Tissue Woundingxe2x80x9d, Plant Cell Physiology, (1 990), pp. 1021-1029; and Nakagawa, et al., xe2x80x9cCloning of a Complementary DNA for Auxin-Induced 1-Aminocyclopropane-1-carboxylate Synthase and Differential Expression of the Gene by Auxin and Woundingxe2x80x9d, (1991) Plant Cell Physiol, 32; 1153-63). Similar differential regulation of ACC synthase gene expression takes place in carnation flowers by wounding, or during senescence (Park, K. Y., et al., xe2x80x9cMolecular cloning of an 1-aminocyclopropane-1-carboxylate synthase from senescing carnation flower petalsxe2x80x9d, Plant Molecular Biology, (1992), Vol. 18, pp. 377-386). The evolution of ACC synthase genes into a multigene family that responds differentially during plant development or in response to stimuli external to the plant (Rottmann, W. H., et al., xe2x80x9cTheologis: A 1-Aminocyclopropane-i-Carboxylate Synthase in Tomato Is Encoded by a Multigene Family Whose Transcription Is Induced During Fruit and Floral Senescencexe2x80x9d, Journal of Molecular Biology, (1991), pp. 937-961) may be a reflection of the importance of ethylene in plants. (See also Slater, A., et al., xe2x80x9cIsolation and characterization of cDNA clones for Tomato polygalacturonase and other ripening related proteinsxe2x80x9d, (1985), Plant Mol Bio vol. 15, pp. 137-147; Smith, T. F. and M. S. Waterman, xe2x80x9cIndentification of Common Molecular Subsequences,xe2x80x9d J. Molecular Biology (1981), pp. 195-197; and Smith, C. J. S., et al., xe2x80x9cAntisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoesxe2x80x9d, Letters to Nature, Nature, (1991), pp. 724-726; Hamilton, A J, et a, xe2x80x9cAntisense Gene that Inhibits Synthesis of the Hormone Ethylene in Transgenic Plantsxe2x80x9d, Nature, (1990), pp. 284-287).
The discovery of the foregoing and of other properties has lead to an understanding that it may be desirable to attempt to genetically alter the production of ethylene in plants. This approach, however, may be considered in some ways delicate. Elimination of ethylene is not a desired result as in many instances it will kill the plant. Modulation of ethylenexe2x80x94at the appropriate timesxe2x80x94is the critical goal, not elimination of it entirely. Modulation of ethylene has been attempted with respect to at least two points in the pathway: 1) the production of ACC in response to ACC synthase, and 2) the oxidation of ACC in response to a different enzyme, ACC oxidase. Because regulation of production of ACC synthase in response to ACC synthase can permit stable modulation and not only total elimination of ethylene, it is a preferred technique. To date, however, successful reduction of the production of ethylene through an alteration at the ACC synthase step in the pathway has only been accomplished in one plant, tomato (Oeller, P. W., et al., xe2x80x9cReversible Inhibition of Tomato Fruit Senescence by Antisense RNAxe2x80x9d, Science, (1991), pp. 437-439). In spite of the seemingly simple conceptual nature of this goal, the actual accomplishment of an alteration of the ethylene biosynthetic pathway through the regulation of ACC synthase production has remained elusive. This is particularly true for the geranium plant, perhaps due to the fact that the identification of full length genes can be difficult for plants. As discussed later, this may, in part, be due to the fact that isolation of full length or high quality RNA has been deemed xe2x80x9cnotoriously difficultxe2x80x9d for plants. (John, M. E., xe2x80x9cAn efficient method for isolation of RNA and DNA from plants containing polyphenolicsxe2x80x9d, Nucleic Acids Research (1992), pp. 2381).
Efforts by others highlight some of the difficulty involved. Recently, Arteca""s laboratory (Wang, T. W. and Arteca, R. N., xe2x80x9cIdentification and Characterization of cDNAs Encoding Ethylene Biosynthetic Enzymes from Pelargonium X Hortorum CV Snow Mass Leavesxe2x80x9d, Plant Physiology, (1995), pp. 627-636) studied two cDNA molecules encoding ACC synthase from a white flower variety of a flowering geranium plant (Pelargonium x hortorum cv Snow Mass Leaves). As their publication explained (perhaps after the fact), these researchers tried to identify and characterize two clones, GAC-1 and GAC-2. In spite of their efforts, they were only able to completely identify one of those cDNA gene sequences, GAC-1. Their study examined the expression of these ACC synthase genes in different plant parts of the geranium and in response to stress induced by osmotic changes (sorbitol) or metal ions (CuCl2). It also evaluated the effects of ethylene on auxin 2,4-D induction in geranium leaves. The study indicated that GAC-1 expression was induced only by stress, whereas expression of GAC-2 appeared to be developmentally regulated. Furthermore, these authors speculated about possible future xe2x80x9ctransfer of antisense GAC-1, GAC-2 . . . into Pelargonium tissues through the Agrobacterium transformation or particle bombardment.xe2x80x9d This confirms a desire in the art for an ACC synthase approach to altering ethylene production in such plants. In spite of this desire, however, the isolation and identification of some, if not all, the ACC synthase gene sequencesxe2x80x94for geranium remained elusive.
Although several plant ACC synthase genes have been identified and sequenced, the current invention describes ACC synthase gene sequences which were previously unknown and which are not believed to have been easily discoverable. As mentioned, one factor which may have militated against an expectation of successfully cloning a plant gene is the particular difficulty in obtaining high-quality and full-length RNA from plants. Indeed, this process has been characterized as xe2x80x9cnotoriously difficultxe2x80x9d by at least more than one practitioner of the art (John, M. E., xe2x80x9cAn efficient method for isolation of RNA and DNA from plants containing polyphenolicsxe2x80x9d, Nucleic Acids Research (1992), pp. 2381 and Logemann, J., et al., xe2x80x9cImproved Method for the Isolation of RNA from Plant Tissuesxe2x80x9d, Analytical Biochem (1987), pp. 16-20)). While this proved to be true for the present inventor, these difficulties were overcome by assessing a new approach to the RNA isolation process. The current inventor, after finding traditional RNA isolation methods to be ineffective, was forced to develop a non-traditional approach described herein. Basically, even though those of ordinary skill in the art had long desired to identify some gene or portion of a gene to manipulate to alter the production of ethylene in some plants, in this case, they failed to realize that the problem lay in the need for a better isolation process. Even though the implementing technology for this process had long been available, those in the art apparently failed to realize how to use that technology to achieve the results now described. To some extent they simply may not have defined the problem, preventing the achievement of the goals sought. Their efforts may properly be characterized as having taught away from the direction taken by the present inventor and, thus, the results achieved here should be considered unexpected.
Difficulties in isolating full-length mRNA in this specific case are also further reflected by the fact that one of the sequences isolated by the current inventor (clone pPHSacc49), though it may bear some similarity to portions of the clone termed GAC-2 by Wang et al., supra, (which, in any case, may have been discovered after the making of the present invention) is actually considerably longer than GAC-2. This highlights the difficulty in successfully isolating a full-length mRNA molecule using standard RNA isolation procedures in certain plant materials. Furthermore, the current inventor has previously isolated a third novel full-length clone (pPHSacc44). Moreover, the high quality RNA (as defined below) isolated by the current inventor is further evidenced by the fact that full length cDNA clones were obtained for pPHSacc44 and pPHSacc49, and all of them could be successfully expressed in an in vitro expression system. In each case, full length ACC synthase (enzyme) protein was synthesized in vitro. In contrast, even later publications by Arteca""s group do not describe the actual in vitro expression of any of the isolated DNA clones.
This is significant because it highlights the difficulty in identifying full length ACC synthase genes. Derivation of DNA encoding ACC synthase from a genomic clone rarely is successful, and therefore, simply would not provide a reasonable expectation of success to one of ordinary skill. Only by utilizing a new and different approach did the present invention successfully identify not only one but several full length ACC synthase gene sequences, and the partial sequence which comprises the instant invention, from the geranium plant. Basically, it was this high quality library containing full length cDNA clones which allowed the present inventor to successfully achieve direct cloning of ACC synthase cDNA. The prior art did not discover these sequences because it could not have: the genes did not exist in the available libraries. It was this new approach which overcame the problems faced, but not solved, by others and resulted in the extraordinary successes described herein. Mere comparison to other genes in the same or different plants did not and could not have yielded the successes described here. The existence of the cDNAs of interest in the library was the governing factor. Thus, even with a viable identification process, successful identification of the several geranium ACC synthase genes, let alone the actual alteration of the plant itself by means of this knowledge, would not have been likely.
Additionally, it should be understood that knowledge of the full DNA sequence of a gene from other plants simply does not lead one to the sequences of the homologous genes in the geranium plant. First, as mentioned earlier, the genes encoding ACC synthase have evolved into a multigene system. There appears to be no single gene, but rather a family of genes in most cases. Thus, knowledge of one gene in one plant species is not certain to lead to one (or several) homologous or analogous genes in another plant species. Second, because known ACC synthase genes are typically so diverse in their nucleotide sequences, knowledge of one would not lead a person of ordinary skill in the art to an expectation of success in isolating the ACC synthase from geranium.
Antisense technology is a well known approach to creating a plant that produces less of a selected protein. Through this technology, a plant is altered by introducing a foreign DNA sequence that encodes an mRNA product complementary to part or all of the plant""s xe2x80x9csensexe2x80x9d mRNA encoding the protein. The presence of antisense RNA inhibits RNA function within a cell (and whole organism). Antisense RNA can bind in a highly specific manner to its complementary sense RNA resulting in blockade in processing or translation of the sense mRNA. Antisense RNA may also disrupt interactions between sense mRNA and sequence-specific RNA binding proteins. Antisense technology may be employed to inhibit the synthesis of an enzyme involved in ethylene biosynthesis. The partial gene sequence identified by the current inventor and disclosed herein may be used for the conception of antisense sequences specific for ACC synthase mRNA. Introduction of the DNA encoding such antisense RNA sequences into a geranium plant results in a plant which may stably produce less ethylene.
The incorporation of antisense RNA in plants as a means to inhibit the synthesis of enzymes has been described by various investigators. Rothstein, et al, xe2x80x9cStable and heritable inhibition of the expression of nopaline synthase in tobacco expressing antisense RNAxe2x80x9d, (1987) Proc. Natl. Acad. Sci. 84: 8439-8443, found that antisense RNA inhibited nopaline synthase (nos) in tobacco.Smith, C. J. S., et al, xe2x80x9cAntisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoesxe2x80x9d, Letters to Nature, Nature, (1991), pp. 724-726, reported that antisense RNA inhibited polygalacturonase in tomato. Others have used antisense RNA to inhibit the synthesis of enzymes involved in ethylene formation. (Oeller, P. W., et al., xe2x80x9cReversible Inhibition of Tomato Fruit Senescence by Antisense RNAxe2x80x9d, Science, (1991), pp. 437-439), expressed RNA antisense to ACC synthase in tomato plants. Others have expressed antisense RNA to a different ethylene forming enzyme (EFE), ACC oxidase, in carnation and tomato. (Michael, M. Z., et al, xe2x80x9cCloning of Ethylene Biosynthetic Genes Involved in Petal Senescence of Carnation and Petunia, and their Antisense Expression in Transgenic Plantsxe2x80x9d, Cellular and Molecular Aspects of the Plant Hormone: Ethylene, (1993), pp. 298-303); Hamilton, A J, et a, xe2x80x9cAntisense Gene that Inhibits Synthesis of the Hormone Ethylene in Transgenic Plantsxe2x80x9d, Nature, (1990), pp. 284-287; Gray, J. E., et al, xe2x80x9cAltered Gene Expression, Leaf Senescence, and Fruit Ripening by Inhibiting Ethylene Synthesis with EFE-Antisense Genesxe2x80x9d, Cellular and Molecular Aspects of the Plant Hormone Ethylene, pp. 82-89, (1993); Murray, A. J. xe2x80x9cExpression of EFE Antisense RNA in Tomato Causes Retardation of Leaf Senescense and Most Fruit Ripening Characteristicsxe2x80x9d, Cellular and Molecular Aspects of the Plant Hormone: Ethylene, (1993), pp. 327-328). The above work with antisense RNA may also be applicable to efforts to stably incorporate the partial gene sequence identified by the current inventor and their antisense sequences into geranium plants. Similarly, the success in expressing antisense RNA for ACC synthase in tomato plants may also be applicable (Oeller, et al., supra). It is noteworthy, and perhaps surprising, that neither of the foregoing disclosures have led to the long sought goal of stably altering ethylene production in geranium plants. Hence, an altered geranium plant expressing reduced levels of ethylene has not been described. The incorporation of ACC synthase antisense DNA into a geranium plant has remained elusive because the complete ACC gene sequences were not available prior to the present invention. The discoveries disclosed herein enable the production of an appropriately altered geranium plant expressing ACC synthase antisense sequences and stably producing reduced levels of ethylene.
The broad object of the invention can be to provide a method for genetic modification of geranium plants (or may be applicable to other plants as well) to control their levels of ethylene. Controlling the level of ethylene in geranium plants may comprise the use of the instant invention individually as disclosed below, or in combination with previously identified genes from geranium or rose for which the full length DNA sequences have also been previously disclosed, or in combination with geranium promoter sequences. See, 1-Aminocyclopropane-1-carboxylate Synthase Genes From Pelargonium, U.S. Pat. No. 5,824,875; 1-Aminocyclopropane-1-Carboxylate Synthase Genes From Pelargonium And Rosa To Control Ethylene Levels In Geraniums And Roses, PCT Patent Application No. PCT/US97/17644; 1-Aminocyclopropane-1-Carboxylate Synthase Genes From Rosa To Control Ethylene Levels In Roses, U.S. National Phase Application No. 09/171,482; and Plant Promoter, U.S. Patent Application No. 60/203021, each hereby incorporated by reference. The instant invention used individually or in combination with such disclosed DNA molecules, fragments thereof, or combinations of such molecules or fragments, may be introduced into a plant cell in reverse orientation to inhibit expression of ACC synthase, thereby reducing the levels of endogenous ethylene.
Another broad object of the invention is to produce transgenic plants which may be monitored for growth and development. Those plants exhibiting prolonged shelf-life with respect to plant growth, flowering, or reduced yellowing of leaves due to reduction in levels of ethylene are to be selected and propagated as premier products with improved properties including reduced leaf yellowing and petal abscission during shipping and storage.
The present invention comprises the identification of the PHSacc-25 gene of geranium which encodes for ACC synthase enzyme or a functional derivative of the gene. The invention further comprises an isolated cDNA molecule having sequence homologous to the PHSacc-25 gene DNA sequence which encodes the ACC synthase enzyme of geranium or a functional derivative.
The invention further comprises the identity of a portion of the isolated cDNA molecule (SEQ ID NO:1).
In another embodiment, the present invention provides the protein encoded by the cDNA molecule as described above, or a functional derivative thereof.
Also provided herein is an antisense oligonucleotide or polynucleotide which may encode an RNA molecule which is complementary to at least a portion of an RNA transcript of the ACC synthase gene described above, which RNA molecule hybridizes with the RNA transcript such that expression of the ACC synthase enzyme is altered.
The above antisense oligonucleotide or polynucleotide molecule can be full length or preferably has between six and 100 nucleotides.
The antisense oligonucleotide or polynucleotide may be complementary to at least a portion of one strand of the cDNA.
An antisense oligonucleotide as described above may be complementary to at least a part of the cDNA sequence SEQ ID NO:1 which part is, for example, from nucleotides 1-50; nucleotides 51-100; nucleotides 101-150; nucleotides 151-200; nucleotides 201-250; nucleotides 251-300; 301-350; or 351-400, and so forth.
In one embodiment, the antisense oligonucleotide can be complementary to at least a part of a 5xe2x80x2 non-coding portion of one strand of the isolated cDNA molecule.
This invention can be further directed to a vector useful for transfection of a geranium plant cell, comprising:
(a) an antisense oligonucleotide or polynucleotide as described above;
(b) regulatory sequences required for expression of the oligonucleotide or polynucleotide in the cell.
The regulatory sequences comprise a promoter active in the cell, which may be an inducible promoter or preferably, a constitutive promoter. The vector preferably further comprise a polyadenylation signal.
In the above vector, the promoter is preferably a heterologous promoter such as a viral promoter. A preferred viral promoter is the CaMV 35S promoter or a promoter homologous to CaMV35S.
In other embodiments, the promoter is selected from the group consisting of the SSU gene promoter, ribulose bisphosphate carboxylase promoter, chlorophyll a/b binding protein promoter, potato ST-LS1 gene promoter, soybean heat shock protein hspl7.5-E promoter, soybean heat shock protein hspl7.3-B promoter, phenylalanine ammonia-lyase promoter, petunia 5-enolpyruvylshikimate-3-phosphate synthase gene promoter, Rhizobium meliloti FIXD gene promoter and nopaline synthase promoter, or the naturally occurring promoter for the geranium ACC synthase gene itself.
The invention can also provide a geranium cell transformed with such a vector as described above, a plantlet or mature geranium plant regenerated from such a cell, or a plant part from such a plant.
The present invention is further directed to a method to alter expression of an ACC synthase enzyme in a geranium cell, plant or a cutting thereof, comprising
(a) transforming a geranium cell or plant with a vector according to any of the prior directions; and
(b) allowing the antisense oligonucleotide or polynucleotide to be expressed and to hybridize with nucleic acid molecules in the cell, plant, or cutting which encode for the ACC synthase enzyme.
Also provided is a method of producing a geranium plant having reduced ethylene production compared to an unmodified geranium plant, comprising the steps of:
(a) transforming a geranium plant with a vector as above;
(b) allowing the plant to grow to at least a plantlet stage;
(c) testing the plant for ACC synthase enzymatic activity or ethylene production; and
(d) selecting a plant having altered ACC synthase activity and/or altered ethylene production compared to an unmodified geranium plant.
A geranium plant produced as above, or progeny, hybrids, clones or plants parts thereof, preferably exhibits reduced ACC synthase expression and reduced ethylene production.
In another embodiment, the invention is directed to a method for producing a geranium variety (or line), characterized by reduced expression or activity of an ACC synthase enzyme and reduced ethylene production compared to an unmodified geranium variety, comprising producing a geranium plant as above and selfing the plant to generate the variety.
Also provided is a method for producing a variant plant of a non-geranium species, an ACC synthase genes of which is homologous to a geranium ACC synthase gene, in which variant plant the ACC synthase expression is altered in comparison to an unmodified plant of the species, comprising
(a) identifying and isolating an ACC synthase gene of the species by hybridization with a sense DNA molecule as described above
(b) constructing a vector which comprises an antisense DNA sequence encoding at least a part of the gene identified in step (a) in an antisense orientation such that
(i) an RNA transcript of the antisense DNA sequence is complementary to the part of the gene, and
(ii) expression of the antisense DNA sequence alters expression of the ACC synthase gene;
(c) transforming a cell of a plant of the species with the vector of step (b) to generate a transformed cell; and
(d) regenerating a plant from the transformed cell of step (c), to produce the variant plant.
The above method is also used to produce a plant variety in a non-geranium plant species characterized by reduced expression or activity of an ACC synthase enzyme and reduced ethylene production compared to a conventional variety of the species, comprising producing a variant plant as above, and selfing the plant to generate the variety.
This invention also provides a method for genetically altering a plant, preferably a plant of a low RNA species, comprising the steps of:
(a) isolating mRNA of the plant using the 2-butoxyethanol precipitation technique wherein at least about 3-5 grams of plant tissue starting material is used to attain a critical mass amount of RNA for precipitation;
(b) constructing a cDNA library from the isolated mRNA
(c) identifying and cloning a desired DNA sequence from the library
(d) genetically altering the cloned DNA sequence;
(e) transforming cells of the plant or the plant directly with the altered DNA sequence; and
(f) if done through a cell-based technique, reproducing a plant from the cells which plant expresses the altered DNA sequence,
thereby genetically altering the plant.
In the above method, the plant is preferably a species of the genus Pelargonium or Rosa, most preferably a geranium plant. In the above method, the cloned DNA sequence preferably encodes ACC synthase.
The above method is used to produce a genetically altered geranium plant, comprising the steps of:
(a) isolating geranium mRNA using a 2-butoxyethanol precipitation technique wherein at least about 3-5 grams of plant tissue starting material is used to attain a critical mass amount of RNA for precipitation;
(b) constructing a cDNA library from the isolated mRNA
(c) identifying and cloning at least one DNA sequence from the library
(d) genetically altering the cloned DNA sequence;
(e) transforming geranium cells with the altered DNA sequence; and
(f) regenerating the genetically altered geranium plant from the cells, which plant expresses the altered DNA sequence.
The invention is further directed to a method of isolating plant mRNA, comprising the steps of:
(a) extracting nucleic acids from a sufficient amount of plant tissue starting material to attain a critical mass amount of RNA for precipitation;
(b) isolating RNA from the nucleic acids of step (a) using a 2-butoxyethanol precipitation technique;
(c) contacting the RNA with a binding partner for mRNA, for example oligo-dT or another molecule or entity which has the characteristics of binding specifically to mRNA with the exclusion of other forms of RNA or DNA. The binding partner may be immobilized on a solid phase or carrier; this yields immobilized mRNA; and
(d) eluting the immobilized mRNA from the carrier by conventional elution methods, or obtaining bound mRNA, thereby isolating the mRNA from total RNA.